Award details

Macromolecular response to pressure

Reference	BB/D521230/1
Principal Investigator / Supervisor	Professor Michael Williamson
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	186,563
Status	Completed
Type	Research Grant
Start date	21/11/2005
End date	20/11/2008
Duration	36 months

Abstract

In a previous BBSRC grant, we have developed a robust method which allows the 1H NMR chemical shift changes on increasing the pressure from 1 bar to 2 kbar to be used as restraints in a molecular dynamics calculation of the change of a protein structure with pressure. The method has been applied on lysozyme and BPTI, and has shown that the helices in both proteins compress fairly uniformly by approximately 1 per cent (with shortening of hydrogen bonds, and a closer approach of adjacent helices), and that the Beta-sheet in lysozyme is almost incompressible, but distorts under pressure. The largest changes in local volume (both positive and negative) are located close to buried water molecules, and it was concluded that buried water molecules play a large part in determining compressibility in proteins. These results are important not just for showing us how proteins behave under pressure, but also because compressibility is proportional to mean volume fluctuation: the results therefore show us which parts of the protein can fluctuate under normal pressure. This provides valuable information for understanding the energetics of protein folding, internal entropy in proteins, and conformational flexibility, which are particularly important when we try to understand enzymes, since internal flexibility is vital for enzyme function. The importance of this study therefore extends far beyond pressure effects. The method has so far only been applied to two proteins, which is not enough to develop any general conclusions. We therefore wish in this proposal to extend the study to two more proteins, Protein G and barnase. These are both well studied proteins, which will allow us to relate our findings to the extensive knowledge base already available. Barnase is an enzyme, and it will be of particular interest to see if the flexibility is concentrated near the active site (as it was for lysozyme), and also to add inhibitors and see how the flexibility is altered. Such a study would be very difficult to do with lysozyme because of the difficulty of labelling lysozyme with 13C and 15N. Our method has so far only been applied to 1H shifts. We propose to extend it to cover 13C shifts as well. This will improve the accuracy of the calculation, but more importantly it will give us access to a nucleus that is more predictably affected by local conformational change. In the longer term it should allow us to develop methods for calculating structural changes resulting from ligand binding, though this is not part of the current proposal. Finally, we also propose to study conformational flexibility in a DNA hairpin (for which we already have the date). This will be valuable information for groups modelling conformational changes in DNA such as bending and base flipping, since it will demonstrate in detail the conformational flexibility of the hairpin. It should also confirm that an AT basepair is more easily compressed (and twisted?) than a GC basepair.

Summary

Proteins are not rigid: they move around all the time. Their motions is very complicated: they have very rapid motions, which are like little wobbling motions, and they also have much larger scale floppy motions, which are slower. We know something about these motions from computer simulations, and something from experiments, but basically we know surprisingly little in detail. These motions are important, because they contribute to the entropy of the protein: the amount of disorder in the protein. The entropy is one of the two main factors contributing to the stability of the protein (the other being hydrogen bonds and other non-covalent bonds in the protein). This proposal aims to measure the scale and location of these motions, by measuring how much the volume fluctuates in different parts of the protein. We will do this by a slightly indirect method: we will carry out experiments to see how NMR spectra change as the protein is put under a high pressure. The changes in the spectra are used to calculate how the structure changes when pressure is applied, and this change is proportional to the volume fluctuation. The calculation of course also tells us directly how the structure changes at high pressure. The experiments will actually be done in Japan (and some have been done already), and use a pressure of 2 kbar, which is roughly twice the pressure found at the very bottom of the ocean. We are planning to carry out calculations on two different proteins, which we already know quite a lot about. It is a deliberate choice to pick well-known proteins, because we can then check that our results agree with what other people have found out about the proteins. We will use two different kinds of experimental information, which are the changes in frequency of the hydrogen and carbon nuclei in the protein as the pressure is increased. One of these proteins is an enzyme, and this will allow us to see how the motions in an enzyme change when it binds to an inhibitor, which will give us important information on how it manages to catalyse reactions. For both proteins we expect to see that interactions with water will be important for controlling flexibility. We will also look how DNA is affected at high pressure. We expect to find that the A-T base pair (which is held together to two hydrogen bonds) is easier to squeeze together than a G-C base pair (which is held together by three hydrogen bonds). It sounds obvious that this should be true, but actually this has not been measured before, and the difference in compressibility will be very useful for calculating how easy it is to distort DNA.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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